SUMMARY Ion channels play an essential role in many biological functions and have been implicated in multiple diseases. As such, there is a need for new potent, bioactive small molecules that can act as ion channel blockers and as probes for biomedical research. Tetracaine is a known small molecule ion channel blocker that binds to diverse molecular targets. The objective of this project is to develop tetracaine derivatives as reversible ion channel antagonists with high affinity, longer lifetimes, and high selectivity for cyclic nucleotide-gated (CNG). The long- term goal is developing an understanding of the structure-activity relationships of tetracaine to design new therapeutics for blinding retinal diseases, such as retinitis pigmentosa (RP). CNG channel blockers have shown great promise for treatment of RP, but there is a critical need for the development of a new generation of blockers with greater selectivity for rod CNG channels and ease of delivery. The project will test the central hypothesis that modifying the three main regions of tetracaine will improve ion channel block by enhancing the affinity, selectivity, and lifetime of the molecule in biological systems. The objective of this project will be accomplished by three specific aims: (1) Tetracaine derivatives with enhanced potency for blocking CNG channels will be synthesized. The tail region will be altered to elucidate the role of the aniline proton as well as to optimize binding. The aromatic core will be modified with electron-withdrawing substituents, both exocyclic and endocyclic, to enhance affinity, selectivity, and water solubility. The effect of modifying the lipophilicity of the tail and the position of substitution on channel binding affinity and selectivity will also be measured. All derivatives will be tested for potency as blockers of human rod and cone CNG channels using patch-clamp electrophysiology following expression in the Xenopus oocyte system. (2) Tetracaine derivatives will be created with increased lifetimes and enhanced hydrolytic stability. To reduce the rate of tetracaine metabolism due to hydrolysis of the ester linkage, tetracaine derivatives with a modified head-linkage group, including an inverted ester and amide head linkage, will be generated to take advantage of enzyme specificity. In an attempt to better understand the role of the carbonyl group, it will be replaced with ether and amine functionalities. The hydrolysis rates will be measured via a butyrylcholinesterase assay. (3) Promising derivatives will be tested for their effects on retinal function using electroretinography following injection into the eye. This will allow us to determine their ability to block light signaling in rod vs. cone photoreceptors and their lifetime within the eye. Finally, derivatives will be tested for their ability to slow the disease progression in a mouse model of RP. This project is designed to enhance the research experience of students at Willamette University who are interested in careers in medicinal chemistry, pharmacology, or health professions. The proposed research will enable them to synthesize novel tetracaine derivative, and then test them at collaborators? labs using patch-clamp electrophysiology or mouse models of retinal disease. These experiences will be invaluable as they embark on their future career path.